The present invention relates to a process for measuring a body fluid component by an enzymatic method using an oxidizable color reagent. More particularly, it relates to a process for measuring a substrate or an enzymatic activity by an enzymatic method using an oxidiazable color reagent which process is free from influences of interfering substances such as bilirubin and hemoglobin which are present in body fluids.
Measurement of living body components, for example, body fluid .components such as blood and urine is indispensable for diagnoses of diseases, elucidation of pathosis, and judgement of progress towards recovery because the change of amounts of the living body components is closely related to diseases. There have been developed methods for measurement of great many body fluid components such as cholesterol, uric acid, glucose, triglycerides, phospholipids, choline, creatine, creatinine, bile acid, monoamine oxidase, etc. It is generally known that these methods are useful for diagnoses of diseases.
As methods for measurement of serum components, so-called "enzymatic methods" have been generalized. In the enzymatic methods, when an objective component is other than enzymes, there is used an enzyme which acts specifically on the objective component. When an objective component is an enzyme, there is used a compound which is a substrate for the enzyme. In each of these cases, enzymatic reaction is carried out and the reaction product is measured, whereby the amount of objective component is determined. Of the enzymatic methods, there are being increased, with development of oxidizable color reagents, methods which comprise acting a hydrogen peroxide producing enzyme, for example, an oxidase, on an objective component or a substance derived therefrom, to produce hydrogen peroxide in an amount corresponding to the amount of the objective component or substance derived therefrom, introducing the hydrogen peroxide into a color-producing system using peroxidase and an oxidizable color reagent as color-producing component, carrying out calorimetric determination on the basis of the coloration thus caused, and thereby determining the amount of the objective component. An example of such method is a method which comprises introducing hydrogen peroxide produced by a combination of cholesterol and cholesterol oxidase; a triglyceride, lipoprotein lipase and glycerol oxidase; uric acid and uricase; or the like into a color-producing system using peroxidase and an oxidizable color reagent, measuring absorbance due to the coloration thus caused, and thereby determining the amount of an objective component.
However, these methods for measuring a body fluid component by an enzymatic method using an oxidizable color reagent tend to be affected by the reducing action of reductive substances which are present in a sample, for example, ascorbic acid, bilirubin, hemoglobin, etc., and therefore negative errors often took place in measured values. Pigments such as hemoglobin and bilirubin cause errors in measurement, depending on measuring wavelength. Moreover, as generally known, absorption due to these pigments themselves varies with the lapse of time during measurement, depending on light, components in reagents, etc. and affects measurement results. Therefore, for removing these interfering substances, various methods have been proposed and investigated.
As methods for decomposing ascorbic acid among the interfering substances, there have been disclosed, for example, a method using ascorbate oxidase (Japanese Patent Publication No. 56-39198), a method using iodic acid or a salt thereof, or periodic acid or a salt thereof (Japanese Patent Appln. Kokai (Laid-Open) Nos. 56-109595, 56-151358 and 56-107161), and a method using copper ions (Japanese Patent Appln. Kokai (Laid-Open) No. 60-262599). Of these methods, the method using ascorbate oxidase in which reaction can be carried out under mild conditions is the most widespread. Since ascorbate oxidase is an enzyme, this method involves an inherent problem, i.e., insufficient heat stability and storage stability, but there has been constituted a measuring system which is hardly affected by ascorbic acid in practice. Therefore, there is a relatively slight desire to seek further improvement in this method. On the other hand, as methods for avoiding the influence of bilirubin, there have been disclosed, for example, a method using copper ions (Japanese Patent Appln. Kokai (Laid-Open) No. 60-262599), a method using a ferrocyanide (Clin. Chem. 26 (20), 227 (1980)), and a method using bilirubin oxidase. These methods involve problems such as insufficient storage stability of reagents, inhibition of an enzyme used in a measuring system and the like, etc., and a satisfactory method has not yet been established. As a method for avoiding the influence of hemoglobin, there has been disclosed, for example, a method using thiourea (Japanese Patent Appln. Kokai (Laid-Open) No. 62-248500). Since thiourea is a strong reducing agent, this method is too disadvantageous to be applied to a measuring system utilizing a redox reaction.